壬基酚对雄性花背蟾蜍抱对、精子及早期发育的影响
摘要
水环境中,壬基酚(Nonylphenol, NP)主要来源于烷基酚聚氧乙烯醚(Alkylphenol ethoxy lates, APE)的降解。APE是一类非离子表面活性剂,广泛用于工业,农业和其他民用领域,而在APE中,应用最广泛的是壬基酚聚氧乙烯醚(Nonylphenol ethoxy lates, NPEO),因此壬基酚成为水环境中主要的环境激素类污染物之一。壬基酚因其具有雌激素效应,在水体中不易降解的特点引起了科研人员的高度重视。本文以繁殖期抱对的雄性花背蟾蜍、花背蟾蜍精子、花背蟾蜍胚胎和蝌蚪为研究对象,分别从体外、体内两方面研究了壬基酚的毒性效应并探讨了其机理,同时对兰州地区水环境中的壬基酚对花背蟾蜍的影响进行了评估。
1、本研究探讨了壬基酚对花背蟾蜍早期胚胎的急性毒性和壬基酚在雄性花背蟾蜍成体内的分布和代谢。结果表明：(1)随着花背蟾蜍的发育,对壬基酚的耐受性有逐渐增强的趋势。壬基酚对22期至39期花背蟾蜍胚胎的24 h小时半致死浓度(Median lethal concentration, LC50)分别为0.18～0.62 mg/L,但35期胚胎的24 h LC50低于26期,说明蟾蜍变态发育过程中各发育期对NP敏感度不同。(2)壬基酚进入花背蟾蜍体内后,可以透过血睾屏障进入睾丸,壬基酚可对花背蟾蜍的睾丸产生直接作用。(3)壬基酚染毒48 h后,雄性蟾蜍肝脏中壬基酚含量增加,血液,睾丸和脑组织中壬基酚含量降低。(4)壬基酚在花背蟾蜍体内可能主要是通过肝代谢。
2、本研究分析了壬基酚对雄性花背蟾蜍抱对行为的影响,并对其机理进行了探讨。研究发现：(1)浓度高于200μg/L壬基酚将会干扰雄性花背蟾蜍的抱对行为,导致抱对蟾蜍分离,而且导致分离的雄性蟾蜍个体死亡。(2)浓度高于200μg/L的壬基酚暴露3d以上将会导致抱对蟾蜍雄性个体血清中的卵泡刺激素和黄体生成素含量显著下降,而对睾酮含量无显著影响。(3)壬基酚暴露导致的卵泡刺激素和黄体生成素含量下降,是壬基酚干扰花背蟾蜍抱对行为的途径之一。
3、以雄性花背蟾蜍和其精子为研究材料,在抱对和受精期间研究了壬基酚对花背蟾蜍精子的影响。研究结果显示：(1)浓度低于400μg/L壬基酚暴露对雄性花背蟾蜍体内的精子动态参数及精子的完整率无显著影响。(2)浓度高于200μg/L的壬基酚直接暴露将显著降低精子活率及其运动速度,并显著降低受精率。(3)精子直接暴露于浓度高于50μg/L壬基酚溶液中将显著增加精子中活性氧(Superoxide dismutase, ROS)水平,而精子中ROS水平与精子活率之间呈高度负相关关系。
4、本研究探讨了壬基酚对花背蟾蜍精子的超微结构的影响。(1)本研究对花背蟾蜍精子的外部形态进行了测量。进一步完善了对于花背蟾蜍精子外部形态的研究数据。(2)壬基酚(≥200μg/L)暴露将会导致花背蟾蜍精子头部、中片和尾部质膜溃散,中片与头部连接处膨胀隆起,其中的线粒体也受到损伤,这可能是导致精子的失活的原因之一
5、本研究探讨了花背蟾蜍卵带胶质对环境污染物吸收的抑制作用。研究表明(1)经过40 h的1 mg/L镉离子和1 mg/L壬基酚暴露实验,结果表明卵带胶质能使胚胎对镉离子及壬基酚的吸收分别降低32.20%和59.03%。(2)胶质对镉离子及壬基酚吸收的抑制效果不同,相对而言胶质对胚胎吸收壬基酚的抑制作用更强。(3)胚胎外胶质能有效降低环境中污染物对胚胎孵化的不利影响。
6、本研究研究了壬基酚对花背蟾蜍早期胚胎发育及蝌蚪体内SOD和MDA的影响。结果表明(1)壬基酚(≥200μg/L)对胚胎发育有明显的阻滞作用,阻滞作用在花背蟾蜍胚胎发育的转动期就已经体现。(2)壬基酚对花背蟾蜍胚胎形态发育的影响主要是缩短了胚胎长度,可能导致胚胎短尾。(3)50μg/L壬基酚暴露25 d,造成花背蟾蜍蝌蚪SOD酶活力的下降及体内(Malonaldehyde, MDA)含量的增加,其影响在短期内(14 d)无法恢复到正常水平。
壬基酚对花背蟾蜍的影响是多方面的。从本研究来看,兰州地区平均环境浓度壬基酚含量对花背蟾蜍的繁殖和早期发育形态均无显著影响。部分排污口水体中的壬基酚含量可能导致花背蟾蜍蝌蚪体内酶活力改变。
Nonylphenol (NP) is the final degradation product of alkylphenol poly ethoxylates (APE), which is a kind of nonionic surfactant widely used in industry, agriculture and other production activities. And, NP is usually present in aquatic environments. Owning to its estrogen effect and high resistance to biodegradation, NP has attracted great attention of researchers. However, as an environmental estrogen widely distributed in water in Lanzhou region, little is known about the effect of NP on the amplexus of amphibia, especially on its sperm and its early development.
The present study mainly investigeted the effects of NP with environmental-relevant concentrations in Lanzhou region on amplexus, sperm and early development of male Bufo raddei. Such information would be valuable for better estimating the risks of NP in relation to the decline of B. raddei around Lanzhou region.
1. The acute toxicity of the NP on the embryo of B. raddei and the distribution of NP in the body of male B. raddei was detected. It was found that:(1) 24 h LC50 of NP of B. raddei was increased gradually during the development of embryo. The 24h LC50 of NP of 22 developing stage to 39 developing stage were 0.18 mg/L to 0.62 mg/L. (2) NP could enter the testes of B. raddei through the blood-testis barrier and the blood-brain barrier. NP could affect the testis tissue directly. (3) The level of NP increased in liver, but decreased in blood, testes and the brain in male B. raddei exposed to NP for 48 h. (4) The mainly tissue of the metabolism of NP in B. raddei was liver.
2. The effects of NP on the amplexus and the hormone of male B. raddei were investigated. The study showed that:(1) NP (≥200μg/L) disturbed the amplexus of male B. raddei, the number of the divorced toad and the death of male B. raddei increased. (2) The level of FSH and LH reduced significantly in male B. raddei exposed to NP (≥200μg/L) for 3 days. And the level of testosterone increased in the blood in male B. raddei exposed to NP (≥50μg/L) for 9 days, but the difference was not significant compared to control. Hence we presume that NP (≥200μg/L) disturbed the amplexus of B. raddei by reducing the level of FSH and LH of male B. raddei.
3. The effects of NP on the sperm of B. raddei during the amplexus and fertilization. The study showed that:(1) NP (≤400μg/L) had no significant effects on the integrity rate or the dynamic parameters of sperm of B. raddei during amplexus. (2) NP (≥200μg/L) significantly reduced the sperm motility and the fertilization rate during fertilization. (3) ROS level significantly increased in the sperm directly exposed to NP (≥50μg/L). And the ROS level had high-negative correlation with the concentration of NP.
4. The effects of NP on the ultra structure of sperm. Results showed that:(1) Present study enriched the data about the ultra structure of the sperm of B. raddei. The sperm of B. raddei was about 58.60μm long, the length of the head and tail of sperm were about 20.47μm and 39.52μm respectively. Sperm was consisted of three parts: a head, a midpiece and a tail. (2) NP (≥200μg/L) seriously impaired the membrane of sperm's head, midpiece and flagellum. The membrane was broken from the head near the midpiece, and the mitochondria were impaired too. The impairment of the structure would lead to the lower sperm motility.
5. The effects of embryo jelly coat to reduce the absorption of pollutants. The results showed that:(1) The 32.20% absorption of Cd2+ and 59.03% absorption of NP of intact embryo were reduced compared to that of dejelly embryo exposed to 1 mg/L Cd2+ or 1 mg/L NP for 40 h. (2) The jelly coat of embryo showed more resistance to NP than the Cd2+. (3) The jelly coat of embryo could reduce and prevent the adverse effects of pollutants on the hatching by reducing the absorbtion.
6. The effects of NP on the morphological development of the embryo. (1) NP (≥200μg/L) blocked the embryo development from the embryonic rotation period. (2) NP (≥200μg/L) would reduce the length of embryo. (3) The decline of SOD and the increase of MDA induced by NP of tadpoles did not return to normal levels within 14 days.
Base on these findings, short-term exposure of B. raddei to NP during amplexus or fertilization in Lanzhou region would not have significant effects on its amplexus, sperm quality, fertilization rate and its early development.
1、本研究探讨了壬基酚对花背蟾蜍早期胚胎的急性毒性和壬基酚在雄性花背蟾蜍成体内的分布和代谢。结果表明：(1)随着花背蟾蜍的发育,对壬基酚的耐受性有逐渐增强的趋势。壬基酚对22期至39期花背蟾蜍胚胎的24 h小时半致死浓度(Median lethal concentration, LC50)分别为0.18～0.62 mg/L,但35期胚胎的24 h LC50低于26期,说明蟾蜍变态发育过程中各发育期对NP敏感度不同。(2)壬基酚进入花背蟾蜍体内后,可以透过血睾屏障进入睾丸,壬基酚可对花背蟾蜍的睾丸产生直接作用。(3)壬基酚染毒48 h后,雄性蟾蜍肝脏中壬基酚含量增加,血液,睾丸和脑组织中壬基酚含量降低。(4)壬基酚在花背蟾蜍体内可能主要是通过肝代谢。
2、本研究分析了壬基酚对雄性花背蟾蜍抱对行为的影响,并对其机理进行了探讨。研究发现：(1)浓度高于200μg/L壬基酚将会干扰雄性花背蟾蜍的抱对行为,导致抱对蟾蜍分离,而且导致分离的雄性蟾蜍个体死亡。(2)浓度高于200μg/L的壬基酚暴露3d以上将会导致抱对蟾蜍雄性个体血清中的卵泡刺激素和黄体生成素含量显著下降,而对睾酮含量无显著影响。(3)壬基酚暴露导致的卵泡刺激素和黄体生成素含量下降,是壬基酚干扰花背蟾蜍抱对行为的途径之一。
3、以雄性花背蟾蜍和其精子为研究材料,在抱对和受精期间研究了壬基酚对花背蟾蜍精子的影响。研究结果显示：(1)浓度低于400μg/L壬基酚暴露对雄性花背蟾蜍体内的精子动态参数及精子的完整率无显著影响。(2)浓度高于200μg/L的壬基酚直接暴露将显著降低精子活率及其运动速度,并显著降低受精率。(3)精子直接暴露于浓度高于50μg/L壬基酚溶液中将显著增加精子中活性氧(Superoxide dismutase, ROS)水平,而精子中ROS水平与精子活率之间呈高度负相关关系。
4、本研究探讨了壬基酚对花背蟾蜍精子的超微结构的影响。(1)本研究对花背蟾蜍精子的外部形态进行了测量。进一步完善了对于花背蟾蜍精子外部形态的研究数据。(2)壬基酚(≥200μg/L)暴露将会导致花背蟾蜍精子头部、中片和尾部质膜溃散,中片与头部连接处膨胀隆起,其中的线粒体也受到损伤,这可能是导致精子的失活的原因之一
5、本研究探讨了花背蟾蜍卵带胶质对环境污染物吸收的抑制作用。研究表明(1)经过40 h的1 mg/L镉离子和1 mg/L壬基酚暴露实验,结果表明卵带胶质能使胚胎对镉离子及壬基酚的吸收分别降低32.20%和59.03%。(2)胶质对镉离子及壬基酚吸收的抑制效果不同,相对而言胶质对胚胎吸收壬基酚的抑制作用更强。(3)胚胎外胶质能有效降低环境中污染物对胚胎孵化的不利影响。
6、本研究研究了壬基酚对花背蟾蜍早期胚胎发育及蝌蚪体内SOD和MDA的影响。结果表明(1)壬基酚(≥200μg/L)对胚胎发育有明显的阻滞作用,阻滞作用在花背蟾蜍胚胎发育的转动期就已经体现。(2)壬基酚对花背蟾蜍胚胎形态发育的影响主要是缩短了胚胎长度,可能导致胚胎短尾。(3)50μg/L壬基酚暴露25 d,造成花背蟾蜍蝌蚪SOD酶活力的下降及体内(Malonaldehyde, MDA)含量的增加,其影响在短期内(14 d)无法恢复到正常水平。
壬基酚对花背蟾蜍的影响是多方面的。从本研究来看,兰州地区平均环境浓度壬基酚含量对花背蟾蜍的繁殖和早期发育形态均无显著影响。部分排污口水体中的壬基酚含量可能导致花背蟾蜍蝌蚪体内酶活力改变。
Nonylphenol (NP) is the final degradation product of alkylphenol poly ethoxylates (APE), which is a kind of nonionic surfactant widely used in industry, agriculture and other production activities. And, NP is usually present in aquatic environments. Owning to its estrogen effect and high resistance to biodegradation, NP has attracted great attention of researchers. However, as an environmental estrogen widely distributed in water in Lanzhou region, little is known about the effect of NP on the amplexus of amphibia, especially on its sperm and its early development.
The present study mainly investigeted the effects of NP with environmental-relevant concentrations in Lanzhou region on amplexus, sperm and early development of male Bufo raddei. Such information would be valuable for better estimating the risks of NP in relation to the decline of B. raddei around Lanzhou region.
1. The acute toxicity of the NP on the embryo of B. raddei and the distribution of NP in the body of male B. raddei was detected. It was found that:(1) 24 h LC50 of NP of B. raddei was increased gradually during the development of embryo. The 24h LC50 of NP of 22 developing stage to 39 developing stage were 0.18 mg/L to 0.62 mg/L. (2) NP could enter the testes of B. raddei through the blood-testis barrier and the blood-brain barrier. NP could affect the testis tissue directly. (3) The level of NP increased in liver, but decreased in blood, testes and the brain in male B. raddei exposed to NP for 48 h. (4) The mainly tissue of the metabolism of NP in B. raddei was liver.
2. The effects of NP on the amplexus and the hormone of male B. raddei were investigated. The study showed that:(1) NP (≥200μg/L) disturbed the amplexus of male B. raddei, the number of the divorced toad and the death of male B. raddei increased. (2) The level of FSH and LH reduced significantly in male B. raddei exposed to NP (≥200μg/L) for 3 days. And the level of testosterone increased in the blood in male B. raddei exposed to NP (≥50μg/L) for 9 days, but the difference was not significant compared to control. Hence we presume that NP (≥200μg/L) disturbed the amplexus of B. raddei by reducing the level of FSH and LH of male B. raddei.
3. The effects of NP on the sperm of B. raddei during the amplexus and fertilization. The study showed that:(1) NP (≤400μg/L) had no significant effects on the integrity rate or the dynamic parameters of sperm of B. raddei during amplexus. (2) NP (≥200μg/L) significantly reduced the sperm motility and the fertilization rate during fertilization. (3) ROS level significantly increased in the sperm directly exposed to NP (≥50μg/L). And the ROS level had high-negative correlation with the concentration of NP.
4. The effects of NP on the ultra structure of sperm. Results showed that:(1) Present study enriched the data about the ultra structure of the sperm of B. raddei. The sperm of B. raddei was about 58.60μm long, the length of the head and tail of sperm were about 20.47μm and 39.52μm respectively. Sperm was consisted of three parts: a head, a midpiece and a tail. (2) NP (≥200μg/L) seriously impaired the membrane of sperm's head, midpiece and flagellum. The membrane was broken from the head near the midpiece, and the mitochondria were impaired too. The impairment of the structure would lead to the lower sperm motility.
5. The effects of embryo jelly coat to reduce the absorption of pollutants. The results showed that:(1) The 32.20% absorption of Cd2+ and 59.03% absorption of NP of intact embryo were reduced compared to that of dejelly embryo exposed to 1 mg/L Cd2+ or 1 mg/L NP for 40 h. (2) The jelly coat of embryo showed more resistance to NP than the Cd2+. (3) The jelly coat of embryo could reduce and prevent the adverse effects of pollutants on the hatching by reducing the absorbtion.
6. The effects of NP on the morphological development of the embryo. (1) NP (≥200μg/L) blocked the embryo development from the embryonic rotation period. (2) NP (≥200μg/L) would reduce the length of embryo. (3) The decline of SOD and the increase of MDA induced by NP of tadpoles did not return to normal levels within 14 days.
Base on these findings, short-term exposure of B. raddei to NP during amplexus or fertilization in Lanzhou region would not have significant effects on its amplexus, sperm quality, fertilization rate and its early development.
引文
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